Natural Killer (NK) lymphocytes do not rearrange or express T cell receptor or immunoglobulin genes and represent 5-15% of peripheral blood mononuclear cells (PBMC). NK cells kill allogeneic cells, infected cells, tumor cells, and some virally infected cells without prior immunization. NK-mediated killing is often inhibited by target cell HLA class I molecules. It has been proposed, that mutations in the HLA class I peptide binding groove can reverse HLA protection from NK-mediated killing. We introduced single point mutations in and around the HLA-B7 peptide binding groove. HLA-B7 variants are expressed in HLA class I negative 721.221 lympohoblasts at similar levels. These cells are used as targets for bulk PBMC NK cells, NK lines and NK clones, generated from 7 healthy human donors. Bulk PBMC show distinct patterns of killing which are largely independent of the donor HLA type. Seven of eleven peptide binding groove mutations allowed significantly increased NK mediated killing, as compared to the unmutated HL-A-B7. Similiarly, four mutations at residues oriented towards the cell receptor also allowed signigficantly increased NK mediated killing. These data suggest that peptide bound to HLA class I as well as surface residues are important for NK recognition. The recognition of the HLA class I by NK cells may be similar to Tcell recognition with regard to the importance of peptide binding groove and surface HLA class I residues. These patterns of recognition are not entirely reproduced by NK lines and clones from the same donors. Our data suggest that although the specificity of NK cells may be broad, clonal distribution of specificities may be selected for in vitro and may depend on the mode of stimulation. Additionally, certain specificities may predominate in bulk PBMC.